WO2021218148A1

WO2021218148A1 - 10-(s)-17-hydrogen-7-dehydro-andrographolidume, industrial chromatography-based preparation method therefor, and use thereof

Info

Publication number: WO2021218148A1
Application number: PCT/CN2020/132794
Authority: WO
Inventors: 刘地发; 郭正友; 王章伟; 张毅; 刘尧奇; 邓双炳; 王振; 方礼; 刘芳芳; 张功俊; 钟仁清
Original assignee: 江西青峰药业有限公司
Priority date: 2020-04-30
Filing date: 2020-11-30
Publication date: 2021-11-04
Also published as: CN113582951A; CN113582951B

Abstract

Provided is a new compound 10-(S)-17-hydrogen-7-dehydro-andrographolidume. An in vitro experiment shows that the compound has a significant inhibition effect on the generation of nitric oxide (NO) in RAW264.7 macrophage induced by lipopolysaccharide (LPS). Therefore, the compound may be used as a nitric oxide inhibitor, and is prepared to an anti-inflammatory drug for treating novel coronavirus pneumonia (COVID-19). Further provided is a preparation method for 10-(S)-17-hydrogen-7-dehydro-andrographolidume; the method is easy to operate and high in yield, can be used for preparing the compound having high purity by using an industrial chromatography technology, and is suitable for large-scale production.

Description

10-(S)-17-hydro-7-dehydroandrographolide and its industrial chromatographic preparation method and use

This application claims the priority of the Chinese patent application 202010362402.1 whose filing date is 2020/4/30. This application quotes the full text of the aforementioned Chinese patent application.

Technical field

The present invention relates to andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide, a preparation method thereof, and use of the andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide for preparing drugs for treating inflammation.

Background technique

Andrographis paniculata (Burm.F.)Nees is extracted from the whole plant of Andrographis paniculata (Burm.F.)Nees. It has antibacterial, anti-inflammatory, detoxification and other effects. It is an important active natural product. Andrographolide and its derivatives have been made into a variety of dosage forms (tablets, dripping pills, capsules, etc.) and are widely used in clinical practice, such as Xiyanping injection, Lianbizhi injection, Yanhuning injection, and Andrographis paniculata Ester tablets, etc. have shown good clinical effects of clearing away heat, detoxifying, anti-bacterial and anti-inflammatory. Luvone et al. believe that nitric oxide (NO) is related to the occurrence of acute and chronic inflammation [Luvone T, Carnuccio R, Di RM. Modulation of granuloma formation endogenous nitric oxide [J]. Eur J Pharmacol, 1994, 265(1/2) ):89-92.]. Andrographolide can significantly down-regulate the expression of inflammatory factors NO, tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in mouse macrophages RAW264.7 induced by lipopolysaccharide, thereby inhibiting the inflammatory response.

At present, Xiyanping injection, which is widely used clinically, has the functions of clearing away heat, detoxifying, antibacterial and anti-inflammatory, but the active ingredient of Xiyanping injection is the sulfonated product of andrographolide, which is not a single structure. The use of single-structure small molecule compounds to further clarify the mechanism is of great significance to the study of its pharmacodynamics and toxicology; although andrographolides have achieved some research results, there is still nothing new for many diseases, especially in the field of anti-inflammatory The particularly effective andrographolide derivatives or analogues are used clinically. Therefore, the development of new andrographolide derivative monomer drugs with anti-inflammatory activity has important social significance.

Summary of the invention

In order to overcome the shortcomings of the prior art, the present invention provides a new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide and its preparation method and application.

The first aspect of the present invention provides a new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide formula (I),

The second aspect of the present invention provides a method for preparing a new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide,

It includes the following steps:

(1) Macroporous adsorption resin column purification: Dissolve andrographolide total sulfonate in water, load the sample on the macroporous adsorption resin column, use a mixed solvent of organic solvent and water for elution, and combine to contain 10-(S)-17- The eluent of hydrogen-7-dehydroandrographolide was concentrated to obtain crude product 1;

(2) Silica gel column purification: dissolve the crude product 1 in a mixed organic solvent (A/B), load the sample on a silica gel column for purification, elution with a mixed organic solvent (A/B), and detect the separation with a UV on-line detector. According to the peak time and chromatographic peak height, determine the start and end time of eluent collection, collect the eluent for HPLC detection, and combine the elution containing 10-(S)-17-hydro-7-dehydroandrographolide according to the test results Liquid, concentrate, get crude product 2;

(3) Recrystallization: Dissolve the crude product 2 in an organic solvent, evaporate part of the organic solvent, and precipitate a solid, filter and dry.

Preferably, in the step (1), the macroporous adsorption resin is a styrene-type macroporous adsorption resin, more preferably, the styrene-type macroporous adsorption resin model HPD-100S, D101S or LX-1180.

Preferably, in the step (1), the organic solvent is an alcohol solvent, such as methanol or ethanol.

Preferably, in the step (1), the organic solvent and water mixed solvent are 0%-60% methanol aqueous solution (preferably 35%-55% methanol aqueous solution) or 0%-60% ethanol aqueous solution (preferably 30%) by volume. %～50% ethanol aqueous solution).

Preferably, in the step (1), the elution is a gradient elution, such as: eluting with a volume ratio of 35%, 45%, and 55% methanol-water solution in sequence, or using a volume ratio of 30%, 40% in sequence. , 50% ethanol-water solution for elution.

Preferably, in the step (1), the concentration temperature is 40-50°C.

Preferably, in the step (2), in the mixed organic solvent (A/B), A is one of petroleum ether or cyclohexane, and B is one of ethyl acetate, dichloromethane, chloroform or acetone ；

More preferably, the mixed organic solvent (A/B) is a petroleum ether/acetone solution;

More preferably, a petroleum ether/acetone solution with a volume ratio of 5:1 to 4:1 is used for dissolution; and/or a petroleum ether/acetone solution with a volume ratio of 5:1 to 1::1 is used for elution.

Preferably, in the step (2), the elution is a gradient elution, for example, a petroleum ether-acetone solution with a volume ratio of 5:1, 3:1, 2:1, and 1:1 is used for elution in sequence.

Preferably, in the step (2), the concentration temperature is 40-50°C.

Preferably, in the step (2), the packing of the HPLC chromatographic column is a reversed-phase C18 packing, more preferably XAqua, ODS-A or ODS-AQ.

Preferably, in the step (2), the detection wavelength of the ultraviolet on-line detector is 150-300 nm, more preferably 225 nm.

Preferably, in the step (3), the organic solvent is ethyl acetate.

Preferably, in the step (3), the number of recrystallization is greater than or equal to 2 times.

Preferably, in the step (3), the drying method is vacuum drying.

Preferably, in the step (3), the drying temperature is 40-50°C.

The third aspect of the present invention provides a new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide as a pharmaceutical active ingredient of the drug formulation, including but not limited to injections, tablets, Capsules, dispersible tablets.

The fourth aspect of the present invention provides the use of the above-mentioned new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide or the above-mentioned pharmaceutical preparation in the preparation of a medicine for the treatment of inflammatory diseases. The inflammatory disease is preferably pneumonia, and the pneumonia is preferably new coronavirus pneumonia (COVID-19).

The beneficial technical effects of the present invention

1. The present invention provides a new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide compound of formula (I). In vitro experiments show that the compound is induced by lipopolysaccharide (LPS) The production of nitric oxide (NO) in RAW264.7 macrophages has a significant inhibitory effect. Therefore, the compound can be used as an inhibitor of nitric oxide and made into an anti-inflammatory drug for the treatment of inflammatory diseases. Further, it can be used for treatment Pneumonia, such as the new coronavirus pneumonia (COVID-19).

2. The preparation method of 10-(S)-17-hydro-7-dehydroandrographolide provided by the present invention has convenient operation and high yield. Industrial chromatography technology can be used to prepare the compound with high purity in large quantities, which is suitable for large-scale production. .

Description of the drawings

Fig. 1: 10-(S)-17-hydro-7-dehydroandrographolide hydrogen nuclear magnetic resonance spectrum of Example 2;

Figure 2: 10-(S)-17-hydro-7-dehydroandrographolide carbon NMR spectrum of Example 2;

Figure 3: HPLC detection diagram of 10-(S)-17-hydro-7-dehydroandrographolide in Example 2;

Figure 4: Example 2 of 10-(S)-17-hydro-7-dehydroandrographolide anti-inflammatory activity test.

Detailed ways

The chemical structural formula of 10-(S)-17-hydro-7-dehydroandrographolide referred to in the following examples (the Arabic number in the structure is the index of the carbon atom in the chemical structure):

Example 1: Preparation of andrographolide total sulfonate

Take 50L of absolute ethanol, place it in the reaction kettle, slowly add 20L of concentrated sulfuric acid, and after stirring evenly, add 50.00kg of andrographolide, stir, and place at room temperature for 72 hours. Control the temperature by adding 50L 95% ethanol, stir, add 50% sodium hydroxide solution to adjust the pH to 7.0, add ethanol to 85% alcohol content, let stand for 24 hours, filter, the filtrate recovers ethanol, concentrates into a thick paste, vacuum dried Get andrographolide total sulfonates.

Example 2-5: Preparation of 10-(S)-17-hydro-7-dehydroandrographolide

Example 2

(1) Purification by macroporous adsorption resin column: Take 200.00g of andrographolide total sulfonate, add appropriate amount of purified water to dissolve it, load the sample on D101S macroporous adsorption resin column, and use volume ratio 30%, 40%, 50% ethanol-water solution in turn Perform elution, combine the eluted fractions containing 10-(S)-17-hydro-7-dehydroandrographolide, and concentrate under reduced pressure at 40°C to obtain crude product 1;

(2) Silica gel column purification: dissolve crude product 1 with an appropriate volume ratio of 5:1 petroleum ether-acetone, then load the sample on a silica gel column, and use petroleum with volume ratios of 5:1, 3:1, 2:1, and 1:1 in turn Ether-acetone solution was used for elution, and the separation situation was detected with an ultraviolet online detector. According to the peak time and the height of the chromatographic peak, the start and end time of the eluent collection was determined, and the eluent was collected for HPLC detection. The HPLC column packing was XAqua (reverse Filling to C18), UV detection wavelength is 225nm. According to the test results, the eluted fractions containing 10-(S)-17-hydro-7-dehydroandrographolide were combined, and concentrated under reduced pressure at 40°C to obtain crude product 2;

(3) Recrystallization: Add appropriate amount of ethyl acetate to dissolve the crude product 2, place it in a fume hood to volatilize part of the solvent until a large amount of solids precipitate, filter, continue to add an appropriate amount of ethyl acetate to dissolve the solid, place a fume hood to volatilize part of the solvent to a large amount of solids precipitate , Filtered, and the solid was dried under reduced pressure at 40°C to obtain 4.53 g of 10-(S)-17-hydro-7-dehydroandrographolide with a purity of 96.33%.

The chemical structure of compound 10-(S)-17-hydro-7-dehydroandrographolide was identified by modern spectroscopy techniques such as NMR and ESI-MS, and the absolute configuration of the compound was determined by X-ray single crystal diffraction. Its physical and chemical properties are as follows:

White powder, molecular formula: C ₂₀ H ₃₀ O ₅ ;

High resolution mass spectrometry HRESIMS m/z 349.2021[M–H] ^– (cald.for.C ₂₀ H ₂₉ O ₅ ,349.2015). The proton nuclear magnetic resonance spectrum ¹ H-NMR (400MH _Z ) and the carbon nuclear magnetic resonance spectrum ¹³ C-NMR (100MH _Z ) are shown in Figure 1 and Figure 2, and the data is shown in Table 1.

Table 1 Hydrogen spectrum and carbon spectrum data of 10-(S)-17-hydro-7-dehydroandrographolide (400/100MHz, DMSO)

Example 3

(1) Purification by macroporous adsorption resin column: Take 200.08g of andrographolide total sulfonate, add appropriate amount of purified water to dissolve it, and load the HPD-100S macroporous resin column with volume ratio of 35%, 45%, and 55% methanol in sequence. The aqueous solution was eluted, and the eluted fractions containing 10-(S)-17-hydro-7-dehydroandrographolide were combined, and concentrated under reduced pressure at 50°C to obtain crude product 1;

(2) Silica gel column purification: Dissolve the crude product 1 in a 5:1 petroleum ether/acetone mixed solvent with a volume ratio of 5:1, and then load the sample on a silica gel column for purification, using volume ratios of 5:1, 3:1, 2:1 in turn 1:1 petroleum ether-acetone solution was used for elution, and the separation situation was detected by UV on-line detector. According to the peak time and chromatographic peak height, determine the start and end time of eluent collection, collect eluent for HPLC detection, HPLC column packing It is ODS-A (reverse C18 filler), and the UV detection wavelength is 225nm. According to the test results, the eluted fractions containing 10-(S)-17-hydro-7-dehydroandrographolide were combined, and concentrated under reduced pressure at 50°C to obtain crude product 2;

(3) Recrystallization: Add appropriate amount of ethyl acetate to dissolve the crude product 2, place it in a fume hood to volatilize part of the solvent until a large amount of solids precipitate, filter, continue to add an appropriate amount of ethyl acetate to dissolve the solid, place a fume hood to volatilize part of the solvent to a large amount of solids precipitate , Filtered, and the solid was dried under reduced pressure at 50°C to obtain 4.39 g of 10-(S)-17-hydro-7-dehydroandrographolide with a purity of 96.54%.

Example 4

(1) Purification by macroporous resin column: Take 200.15g of andrographolide total sulfonate, add appropriate amount of purified water to dissolve it, and load LX-1180 macroporous resin column with 30%, 40%, 50% volume ratio in turn Ethanol-aqueous solution was used for elution, and the eluted fractions containing 10-(S)-17-hydro-7-dehydroandrographolide were combined and concentrated under reduced pressure at 40°C to obtain crude product 1;

(2) Silica gel column purification: Dissolve the crude product 1 with an appropriate volume ratio 4:1 petroleum ether/acetone solution and load the sample on a silica gel column for purification, using volume ratios of 5:1, 3:1, 2:1, 1 :1 petroleum ether-acetone solution for elution, use UV online detector to detect the separation, determine the start and end time of eluent collection according to the peak time and chromatographic peak height, collect eluent for HPLC detection, HPLC column packing It is ODS-AQ (reverse C18 filler), and the UV detection wavelength is 225nm. According to the test results, the eluted fractions containing 10-(S)-17-hydro-7-dehydroandrographolide were combined, and concentrated under reduced pressure at 40°C to obtain crude product 2;

(3) Recrystallization: Add appropriate amount of ethyl acetate to dissolve the crude product 2, place it in a fume hood to volatilize part of the solvent until a large amount of solids precipitate, filter, continue to add an appropriate amount of ethyl acetate to dissolve the solid, place a fume hood to volatilize part of the solvent to a large amount of solids precipitate , Filtered, and the solid was dried under reduced pressure at 50°C to obtain 4.72 g of 10-(S)-17-hydro-7-dehydroandrographolide with a purity of 96.19%.

Example 5

(1) Purification by macroporous adsorption resin column: Take 20.15 kg of andrographolide total sulfonate, add appropriate amount of purified water to dissolve it, and load the HPD-100S macroporous resin column with volume ratio of 35%, 45%, and 55% methanol in sequence. The aqueous solution was eluted, and the eluted fractions containing 10-(S)-17-hydro-7-dehydroandrographolide were combined, and concentrated under reduced pressure at 50°C to obtain crude product 1;

(2) Purification by industrial chromatography-grade silica gel column: Dissolve crude product 1 in a 5:1 petroleum ether/acetone mixed solvent with an appropriate volume ratio and load the sample on an industrial chromatography system for purification (DAC-HB300 dynamic axial compression column, silica gel packing), and then Elute with a volume ratio of 5:1, 3:1, 2:1, 1:1 petroleum ether-acetone solution, use an ultraviolet online detector to detect the separation, and determine the eluent according to the peak time and chromatographic peak height Collect the start and end time, collect the eluent for HPLC detection, the HPLC column packing is ODS-A (reverse C18 packing), and the ultraviolet detection wavelength is 225nm. According to the test results, the eluted fractions containing 10-(S)-17-hydro-7-dehydroandrographolide were combined, and concentrated under reduced pressure at 40°C to obtain crude product 2;

(3) Recrystallization: Add appropriate amount of ethyl acetate to dissolve the crude product 2, place it in a fume hood to volatilize part of the solvent until a large amount of solids precipitate, filter, continue to add an appropriate amount of ethyl acetate to dissolve the solid, place a fume hood to volatilize part of the solvent to a large amount of solids precipitate , Filtered, and the solid was dried under reduced pressure at 40°C to obtain 415.28 g of 10-(S)-17-hydro-7-dehydroandrographolide with a purity of 96.08%.

Example 6: 10-(S)-17-hydro-7-dehydroandrographolide anti-inflammatory activity test

The compound obtained in Example 2 was applied to lipopolysaccharide (LPS)-induced RAW264.7 mouse macrophages, and the level of NO in the culture supernatant was detected by the Griess reagent colorimetric method. The ability of the tested drug to inhibit the release of NO was used as Screen indicators to evaluate the anti-inflammatory activity of the compound.

1. Preparation of drug solution

The monomer compound was dissolved in DMSO to prepare a solution of 1.564, 3.125, 6.250, 12.50, 25.00 μg/ml.

2. Test method

(1) a single cell suspension of mouse macrophages with 10% fetal bovine serum containing culture medium formulated, taking the number of macrophage RAW264.7 mouse seeded into 96-well plates, ¹⁰⁵ cells per well Count and set 3 multiple holes.

(2) After 24 hours of incubation, add different concentrations of test drug to each well and add 1ug/mL LPS at the same time.

(3) After 24 hours of incubation at 37°C, add 100ul Griess solution to each well and continue to incubate for 5 minutes, then stop the incubation.

(4) Measure the optical density (OD) value at 570nm wavelength with an enzyme-linked immunoassay, and calculate the NO inhibition rate.

3. Experimental results

The inhibitory effect of 10-(S)-17-hydro-7-dehydroandrographolide compound on NO in RAW264.7 cells has an IC50 of 9.184 μg/ml, see Figure 4 for details.

The results show that 10-(S)-17-hydro-7-dehydroandrographolide compound has a significant inhibitory effect on the production of RAW264.7NO in mouse macrophages induced by lipopolysaccharide (LPS), and can be used as a nitric oxide inhibitor , Indicating that the compound of the present invention has obvious anti-inflammatory activity; therefore, it can be used to prepare new anti-inflammatory active drugs; further, it can be used to treat pneumonia, such as novel coronavirus pneumonia (COVID-19).

Claims

A new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide, the structure is formula (Ι),
A new preparation method of andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide,

It is characterized in that it includes the following steps:

(1) Macroporous adsorption resin column purification: Dissolve andrographolide total sulfonate in water, load the sample on the macroporous adsorption resin column, use a mixed solvent of organic solvent and water for elution, and combine to contain 10-(S)-17- The eluent of hydrogen-7-dehydroandrographolide was concentrated to obtain crude product 1;

(2) Silica gel column purification: dissolve the crude product 1 in a mixed organic solvent (A/B), load the sample on a silica gel column for purification, elution with a mixed organic solvent (A/B), and detect the separation with a UV on-line detector. According to the peak time and chromatographic peak height, determine the start and end time of eluent collection, collect the eluent for HPLC detection, and combine the elution containing 10-(S)-17-hydro-7-dehydroandrographolide according to the test results Liquid, concentrate, get crude product 2;

(3) Recrystallization: Dissolve the crude product 2 in an organic solvent, evaporate part of the organic solvent, and precipitate a solid, filter, and dry.
The preparation method according to claim 2, characterized in that: in the step (1), the macroporous adsorption resin is a styrene-type macroporous adsorption resin, preferably the resin model HPD-100S, D101S or LX-1180.
The preparation method according to claim 2, characterized in that: in the step (1), the organic solvent is an alcohol solvent; and/or the mixed solvent of the organic solvent and water is a methanol-water solution with a volume ratio of 0% to 60% Or, the volume ratio is 0%-60% ethanol-water solution; and/or, the concentration temperature is 40-50°C.
The preparation method according to claim 2, characterized in that: in the step (2),

In the mixed organic solvent (A/B), A is petroleum ether or cyclohexane, and B is ethyl acetate, dichloromethane, chloroform or acetone;

Preferably, the mixed organic solvent (A/B) is a petroleum ether/acetone solution;

More preferably, a petroleum ether/acetone solution with a volume ratio of 5:1 to 4:1 is used for dissolving; and/or a petroleum ether/acetone solution with a solvent of 5:1 to 1:1 by volume is used for elution.
The preparation method according to claim 2, characterized in that: in the step (2), the packing of the HPLC chromatographic column is a reversed-phase C18 packing, preferably XAqua, ODS-A or ODS-AQ.
The preparation method according to claim 2, characterized in that: in the step (2), the detection wavelength of the ultraviolet on-line detector is 150-300 nm; and/or the concentration temperature is 40-50°C.
The preparation method according to claim 2, characterized in that: in the step (3), the organic solvent is ethyl acetate; and/or, the number of recrystallizations is greater than or equal to 2; and/or, the drying method is reduced Press dry.
A new andrographolide derivative represented by formula (I) as claimed in claim 1, 10-(S)-17-hydro-7-dehydroandrographolide or any one of claims 2-8 The new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide prepared by the method is used as a pharmaceutical preparation of the active ingredient of the medicine. The pharmaceutical preparation preferably includes, but is not limited to, injection, Tablets, capsules, dispersible tablets.
The new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide of claim 1 or the new andrographolide derivative prepared by the method of any one of claims 2-8 Use of 10-(S)-17-hydro-7-dehydroandrographolide or the pharmaceutical preparation of claim 9 in the preparation of a medicine for the treatment of inflammatory diseases, the inflammatory disease is preferably pneumonia, and the pneumonia is preferably a novel coronavirus Pneumonia (COVID-19).